Extraocular Muscle Fibers Research Articles

Complications of Cosmetic Wide Conjunctivectomy Combined With Postsurgical Mitomycin C Application

To raise awareness among clinicians about the risk of severe complications after cosmetic conjunctivectomy plus postsurgical topical mitomycin C (MMC) treatment. We retrospectively reviewed the medical records of 48 consecutive patients who received cosmetic wide conjunctivectomy plus postsurgical topical MMC treatment to eliminate conjunctival injection and visited our outpatient clinic between January 2009 and January 2011. Forty-four patients (91.67%) developed complications after cosmetic wide conjunctivectomy plus postsurgical topical MMC treatment. Complications included chronic conjunctival epithelial defects (22 patients), scleral thinning with (19 patients) or without (2 patients) calcified plaques, fibrovascular conjunctival adhesion at the muscle insertion site (15 patients), chronic dysfunctional tear syndrome (11 patients), avascular zones (10 patients), abnormal vessel growth (5 patients), lymphangiectasis (3 patients), adhesions of Tenon capsule and the conjunctiva at the extraocular muscle insertion site (1 patient), extraocular muscle fiber exposure (1 patient), and diplopia (9 patients). The mean time between surgery and the onset of diplopia was 1.67 months (range, 1-3 months), and the mean deviation was 11.0 prism diopters (range, 0-25 prism diopters) in the primary position. Three patients underwent strabismus surgery due to incomitant esotropia. Eight patients underwent calcified plaque removal because of severe progression of the calcification. Five patients underwent conjunctival flap operation because of severe progressive scleral thinning. Cosmetic wide conjunctivectomy, which has been advertised as a simple and effective surgical option for the treatment of conjunctival injection, has caused disruption in the normal physiology of the ocular surface, which leads to various devastating complications.

Binocular coordination of eye movements – Hering’s Law of equal innervation or uniocular control?

The neurophysiological basis for binocular control of eye movements in primates has been characterized by a scientific controversy that has its origin in the historical conflict of Hering and Helmholtz in the 19th century. This review focuses on two hypotheses, linked to that conflict, that seek to account for binocular coordination - Hering's Law vs. uniocular control of each eye. In an effort to manage the length of the review, the focus is on extracellular single-unit studies of premotor eye movement cells and extraocular motoneurons. In the latter half of the 20th century, these studies provided a wealth of neurophysiological data pertaining to the control of vergence and conjugate eye movements. The data were initially supportive of Hering's Law. More recent data, however, have provided support for uniocular control of each eye consistent with Helmholtz's original idea. The controversy is far from resolved. New anatomical descriptions of the disparate inputs to multiply and singly innervated extraocular muscle fibers challenge the concept of a 'final common pathway' as they suggest there may be separate groups of motoneurons involved in vergence and conjugate control of eye position. These data provide a new challenge for interpretation of uniocular premotor control networks and how they cooperate to produce coordinated eye movements.

Expression of Slow Tropomyosin (TM-) in Fast Fibers of Extraocular Muscles

We previously reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of domestic dog extraocular muscles (EOM) (Bicer & Reiser, Invest. Ophthalmol. Vis. Sci. 45:138-143, 2004 and 50:157-167, 2009). In addition, many dog extraocular fibers, especially from the muscle orbital layer, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. The results of these studies also suggested possible differences in the tropomyosin (TM) isoform composition of fast fibers between the two EOM layers, based upon gel electrophoretic mobility patterns. The objective of this study was to determine whether differences in TM isoform expression exist between fast global and fast orbital fibers in dog EOMs. TM isoforms in global and orbital single fibers were identified by SDS-PAGE and immunoblotting. Fast and slow fibers in the global layer express fast TM-α and slow TM-γ, respectively, plus TM-β, identical to fast and slow fibers in limb muscles. Slow orbital fibers express TM-β and TM-γ, as expected. Fast orbital fibers, on the other hand, have an unusual pattern of TM-γ, along with TM-β and TM-α. The same TM isoform expression patterns were found in Sprague Dawley rat EOM fibers. Co-expression of all three TM isoforms was not observed in single fibers in limb muscles of either species. These results contribute to the understanding of the elaborate diversity in contractile protein isoform expression among mammalian EOM fibers. Supported by the National Science Foundation.

Disorders of vergence eye movements

The aim is to re-interpret disorders of vergence in the light of recent studies that view disjunctive eye movements as but one component of three-dimensional gaze control. Most natural eye movements combine vergence with saccades, pursuit and vestibular eye movements. Electrophysiological studies in epileptic patients, as well as evidence from monkeys, indicate that frontal and parietal cortex govern vergence as a component of three-dimensional gaze. Clinicians apply Hering's law of equal innervation to interpret disjunctive movements as the superposition of conjugate and vergence commands. However, electrophysiological studies indicate that disjunctive saccades are achieved by programming each eye's movement independently. Patients with internuclear ophthalmoplegia (INO) may have preserved vergence, which can be recruited to compensate for loss of conjugacy. Vergence may also enable gaze shifts in saccadic palsy. Some forms of nystagmus suppress or change with convergence; co-contraction of the horizontal rectus muscles does not appear to be the explanation. Rather, effects of near viewing on central vestibular mechanisms or differential activation of specific types of extra-ocular muscle fiber may be responsible. Interpretation of disorders of vergence is aided by applying a scheme in which their contributions to three-dimensional gaze control is considered.

Compartmentalized innervation of primate lateral rectus muscle.

Skeletal and craniofacial muscles are frequently composed of multiple neuromuscular compartments that serve different physiological functions. Evidence of possible regional selectivity in LR intramuscular innervation was sought in a study of the anatomic potential of lateral rectus (LR) muscle compartmentalization. Whole orbits of two humans and five macaque monkeys were serially sectioned at 10-microm thickness and stained with Masson trichrome. The abducens nerve (CN6) was traced anteriorly from the deep orbit as it branched to enter the LR and arborized among extraocular muscle (EOM) fibers. Three-dimensional reconstruction was performed in human and monkey orbits. Findings were in concordance in the monkey and human orbits. External to the LR global surface, CN6 bifurcated into approximately equal-sized trunks before entering the global layer. Subsequent arborization showed a systematic topography, entering a well-defined inferior zone 0.4 to 2.5 mm more posteriorly than branches entering the largely nonoverlapping superior zone. Zonal innervation remained segregated anteriorly and laterally within the LR. Consistent segregation of intramuscular CN6 arborization in humans and monkeys suggests functionally distinct superior and inferior zones for the LR. Since the LR is shaped as a broad vertical strap, segregated control of the two zones could activate them separately, potentially mediating previously unappreciated but substantial torsional and vertical oculorotary LR actions.

Mitochondrial DNA Defects and Selective Extraocular Muscle Involvement in CPEO

PURPOSE. Chronic progressive external ophthalmoplegia (CPEO) is a prominent, and often the only, presentation among patients with mitochondrial diseases. The mechanisms underlying the preferential involvement of extraocular muscles (EOMs) in CPEO were explored in a comprehensive histologic and molecular genetic study, to define the extent of mitochondrial dysfunction in EOMs compared with that in skeletal muscle from the same patient. METHODS. A well-characterized cohort of 13 CPEO patients harboring a variety of primary and secondary mitochondrial (mt)DNA defects was studied. Mitochondrial enzyme function was determined in EOM and quadriceps muscle sections with cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) histochemistry, and the mutation load in single muscle fibers was quantified by real-time PCR and PCR-RFLP assays. RESULTS. CPEO patients with mtDNA deletions had more COX-deficient fibers in EOM (41.6%) than in skeletal muscle (13.7%, P > 0.0001), and single-fiber analysis revealed a lower mutational threshold for COX deficiency in EOM. Patients with mtDNA point mutations had a less severe ocular phenotype, and there was no significant difference in the absolute level of COX deficiency or mutational threshold between these two muscle groups. CONCLUSIONS. The more pronounced mitochondrial biochemical defect and lower mutational threshold in EOM compared with skeletal muscle fibers provide an explanation of the selective muscle involvement in CPEO. The data also suggest that tissue-specific mechanisms are involved in the clonal expansion and expression of secondary mtDNA deletions in CPEO patients with nuclear genetic defects.

Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles

The mammalian genome contains three ancient sarcomeric myosin heavy chain (MYH) genes, MYH14/7b, MYH15 and MYH16, in addition to the two well characterized clusters of skeletal and cardiac MYHs. MYH16 is expressed in jaw muscles of carnivores; however the expression pattern of MYH14 and MYH15 is not known. MYH14 and MYH15 orthologues are present in frogs and birds, coding for chicken slow myosin 2 and ventricular MYH, respectively, whereas only MYH14 orthologues have been detected in fish. In all species the MYH14 gene contains a microRNA, miR-499. Here we report that in rat and mouse, MYH14 and miR-499 transcripts are detected in heart, slow muscles and extraocular (EO) muscles, whereas MYH15 transcripts are detected exclusively in EO muscles. However, MYH14 protein is detected only in a minor fibre population in EO muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles. MYH15 protein is present in most fibres of the orbital layer of EO muscles and in the extracapsular region of bag fibres. During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth. In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles. The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals.

FP34-WE-01 Evidence for selective sparing of fast extraocular muscle fibers in ocular myasthenia gravis

FP34-WE-01 Evidence for selective sparing of fast extraocular muscle fibers in ocular myasthenia gravis

Changes in Dynamic and Kinematic Properties of Saccades in Ocular Myasthenia following Intravenous Immunoglobulin Treatment

The dynamics and kinematics of saccades in a patient with severe ocular myasthenia were studied before and after treatment with intravenous immunoglobulin (IVIG). Before therapy, horizontal saccades were hypometric, but faster than similar-sized saccades made by normal subjects. During a 5-minute test period, saccades decreased in size (fatigue effect), but remained faster than those of controls. Listing's plane of the eye with greater ophthalmoplegia was increased in thickness. After IVIG treatment, the range of eye movements improved, but saccades remained faster than those of controls. Also, no fatigue was observed and the thickness of Listing's plane was reduced toward the normal range. Increased peak velocity, despite progressive hypometria due to fatigue, supports the hypothesis that the pale global extraocular muscle fibers are relatively spared in myasthenia. Involvement of other extraocular muscle-fiber types leads to limited range of eye movements and an increase in the thickness of Listing's plane.

Effects of botulinum a toxin injection on the extraocular muscle fiber layers: Comparison between subtenon injection and intramuscular injection

To compare the morphological changes following injection of botulinum A toxin to the extraocular muscle fiber layers with those following injection to the subtenon intramuscular system. Twelve New Zealand white rabbits were divided into two groups. In the first group (six rabbits), four received injections of 10 units of botulinum A toxin into the subtenon space of the superior rectus muscle OD, and the same dose of botulinum A toxin was injected directly into the superior rectus muscle OS. The other two rabbits in the first group were labeled as control animals; 0.1 ml of normal saline was injected into the subtenon space OD, and direct intramuscular injection was performed on the opposite eye. The animals in the first group were killed after 4 weeks to measure the average diameter of the muscle fibers in both the orbital and global layer. In animals of the second group (six rabbits), the same procedures were performed and the animals were killed 12 weeks after treatment. The average diameter of muscle fibers in both the orbital layer and global layer was markedly reduced in all of the botulinum A toxin-injected groups at 4 weeks after treatment compared with the controls. There was no difference in the average diameter of the muscle fibers between the orbital layer of the subtenon-injected group and that of the intramuscular-injected group at 4 weeks after treatment, but the average diameter of the muscle fibers in the global layer was significantly reduced in the intramuscular-injected group compared with the subtenon-injected group at 4 weeks after treatment. At 12 weeks following treatment, there was no difference in the average diameter between the botulinum A toxin-injected group and the control group regardless of where the toxin was injected. Subtenon injection of botulinum A toxin induced similar morphological changes as direct intramuscular injection in the extraocular muscle fiber layers. These results suggest the possibility of clinical applications of subtenon botulinum A toxin injection for the treatment of strabismus.

The Changes in Extraocular Muscles in Horner's Syndrome of Rats

Purpose: The aim of this study was to understand the pathogenesis of the enophthalmos of Horner's syndrome. Methods: We performed right cervical sympathectomy in 10 Sprague-Dawley rats weighing 200300 grams at 8 weeks of age. We obtained bilateral extraocular muscle and fat from all 10 rats. These tissues were observed under light and electron microscopy. Results: After 8 weeks, ptosis and enophthalmos were seen in the right eye in all rats. We found no change bilateral fat, but the right extraocular muscle fibers had smaller diameters than the left. The right intermuscular distance was longer than the left upon light microscopy and the right extraocular muscle contained fewer and smaller mitochondria than the left extraocular muscle upon electron microscopy. Conclusions: Based on these results, it is believed that enophthalmos resulted from extraocular muscle atrophy that resulted from decreasing metabolism of mitochondria in Horner's syndrome. However, enophthalmos in Horner's syndrome requires more intensive study.

The Changes in Myosin Heavy Chain Isoforms After Extraocular Muscle Recession in Rabbits

Purpose: To study the changes in the amount and isoform pattern of the myosin heavy chain (MyHC) in rabbit extraocular muscle (EOM) fibers after recession. Methods: Sixteen New Zealand white rabbits were used. Recession surgery was performed on the right superior rectus (SR) muscle by 3 mm in eight rabbits, and performed by 8 mm in other eight rabbits. The left SR muscles were left intact as the control groups. The SR muscles in both eyes were harvested from two rabbits from each recession group at 3 days and 1, 2, and 4 weeks after surgery. The changes in MyHC amount and isoform pattern were analyzed by gel electrophoresis. Results: Total MyHC content decreased from 1 week after surgery in the 3-mm recessed group and from 3 days in the 8-mm group. The type IIb MyHC (MyHCIIb) plus EOM-specific MyHC (MyHCeom) showed similar proportional changes to the total MyHC at the different time points after surgery. Conclusions: The fast MyHCIIb plus the superfast MyHCeom decreased after EOM recession, and these results appear to be related to the changes in the global layer rather than in the orbital one. This suggests that the global layer might be the fast and the superfast twitch portions of rabbit EOM, which perform the fast saccades in ocular movements.

Diagnosing disconjugate eye movements

Saccades are fast eye movements that conjugately shift the point of fixation between distant features of interest in the visual environment. Several disorders, affecting sites from brainstem to extraocular muscle, may cause horizontal saccades to become disconjugate. Prior techniques for detection of saccadic disconjugacy, especially in internuclear ophthalmoparesis (INO), have compared only one point in abducting vs adducting saccades, such as peak velocity. We applied a phase-plane technique that compared each eye's velocity as a function of change in position (normalized displacement) in 22 patients with disease variously affecting the brainstem reticular formation, the abducens nucleus, the medial longitudinal fasciculus, the oculomotor nerve, the abducens nerve, the neuromuscular junction, or the extraocular muscles; 10 age-matched subjects served as controls. We found three different patterns of disconjugacy throughout the course of horizontal saccades: early abnormal velocity disconjugacy during the first 10% of the displacement in patients with INO, oculomotor or abducens nerve palsy, and advanced extraocular muscle disease; late disconjugacy in patients with disease affecting the neuromuscular junction; and variable middle-course disconjugacy in patients with pontine lesions. When normal subjects made disconjugate saccades between two targets aligned on one eye, the initial part of the movement remained conjugate. Along with conventional measures of saccades, such as peak velocity, phase planes provide a useful tool to determine the site, extent, and pathogenesis of disconjugacy. We hypothesize that the pale global extraocular muscle fibers, which drive the high-acceleration component of saccades, receive a neural command that ensures initial ocular conjugacy.

The extraocular muscles in Amyotrophic Lateral Sclerosis (ALS)?

Abstract Purpose To investigate the morphology, fiber type composition, contractile proteins, extracelluar matrix (ECM), innervation and capillarisation, of the extraocular muscles (EOM) of patients deceased with ALS, in order to evaluate the possible involvement of EOMs in this disease. Methods EOM and limb muscle samples obtained from 5 ALS patients and age matched controls at autopsy were processed for immunocytochemistry, with monoclonal antibodies against myosin heavy chain isoforms; laminin chains (Ln) α1, α2, α4, α5 and β2; etc. Hematoxylin and eosin, NADH‐TR, acetylcholinesterase and alpha‐bungarotoxin were also used. Results The EOMs of ALS patients appeared remarkably unaffected, in strong contrast to the limb muscles. Wider variation in fiber diameter than normally seen in EOMs, including apparently atrophic and hypertrophic groups of fibers, was noted. MyHC embryonic was only present in sporadic fibers and the pattern of distribution of MyHC slow‐tonic was also affected. In most ALS cases, Ln α4, α5 and β2 isoforms were lost from the basement membrane of the EOM fibers . The laminin content of the neuromuscular junctions was altered, Ln α2 and β2 isoforms were missing in perineurium and endoneurium and Ln α5 was absent in endoneurium. In capillary and blood vessels, Ln α4, α5 were maintained whereas Ln β2 was absent. Conclusion The changes observed in EOMs with ALS suggest that they are not completely spared in this motoneuron disease although they are significantly less affected that limb muscles. The altered contractile protein content and structure‐specific changes in the composition of the ECM are likely to be functionally relevant. Further studies are underway to determine the functional implications of our findings.

Elevated expression in situ of selectin and immunoglobulin superfamily type adhesion molecules in retroocular connective tissues from patients with Graves' ophthalmopathy.

Activation of certain adhesion molecules within vascular endothelium and the surrounding extravascular space is a critical event in the recruitment and targeting of an inflammatory response or autoimmune attack to a particular tissue site. We have recently demonstrated that the adhesion of lymphocytes to cultured retroocular fibroblasts obtained from patients with Graves' ophthalmopathy (GO) is mediated predominantly by the interaction of lymphocyte function-associated antigen-1 (LFA-1), expressed on lymphocytes, with intercellular adhesion molecule-1 (ICAM-1), expressed by these cells following exposure to interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), IL-1 alpha or purified thyroid-stimulating immunoglobulins. We now report the expression and localization in situ of several adhesion molecules, ICAM-1, endothelial leucocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and LFA-3 in retroocular tissues derived from patients with severe GO (n = 4) and normal individuals (n = 3). Serial cryostat sections of tissue specimens were processed for immunoperoxidase staining using various MoAbs against ICAM-1, ELAM-1, VCAM-1 and LFA-3. In addition, consecutive sections were stained with MoAbs against LFA-1, CD45RO (UCHL-1)DR-human leucocyte antigen (HLA-DR), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150,95). In GO-retroocular tissues, strong immunoreactivity for ICAM-1 and LFA-3 was detected in blood vessels (> 90%), in perimysial fibroblasts surrounding extraocular muscle fibres, and in connective tissue distinct from extraocular muscle. No ICAM-1 or LFA-3 immunoreactivity was present in extraocular muscle cells themselves. ICAM-1 and LFA-3 immunoreactivity in normal tissues was minimal or absent both in connective and muscle tissues. Vascular endothelium was strongly positive for ELAM-1 and VCAM-1 in GO-retroocular tissues, while VCAM-1 immunoreactivity was minimal (< 5% of blood vessels) and ELAM-1 immunoreactivity was generally absent in normal retroocular tissue. LFA-1-expressing, activated mononuclear cells and memory T lymphocytes (CD3+/CD45RO+) were only detected in GO-retrocular tissues, and were mainly localized around blood vessels and in areas of ICAM-1-expressing connective and perimysial tissue. HLA-DR expression was restricted to GO-tissue specimens, with strong immunoreactivity detected in blood vessels, macrophages and connective tissue and perimysial fibroblasts. No HLA-DR was detectable in extraocular muscle cells. In conclusion, infiltration of the orbit in GO by mononuclear cells, and their targeting within the orbit, may depend upon the coordinate expression of certain adhesion and MHC molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific Metabolic Properties of Rat Oculorotatory Extraocular Muscles Can Be Linked to Their Low Force Requirements

To gain insight into the metabolic pathways of oculorotatory extraocular muscle (EOM) fiber types at the cellular level to explain their high fatigue resistance, rapid contraction, and low force output. In consecutive sections of adult rat EOMs, the cross-sectional area (CSA) was calculated, and the activities of succinate-dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPDH) were measured by quantitative histochemistry of different fiber types classified by the myofibrillar adenosine triphosphatase (mATPase) staining pattern. In the orbital regions, type 1 (fast) fibers were present, showing small CSA, medium SDH, and low GPDH activity. The type 2 (slow) fibers exhibited extremely small CSA and low SDH and GPDH activity. In the global region, fast types 3 to 5 fibers were found, forming a continuum with an inverse correlation between CSA and enzyme activity. SDH and GPDH activity showed an unusual positive relationship in contrast to the skeletal muscle fiber types. The type 6 fibers were slow forming a close and clearly separated group with medium CSA and extremely low SDH and low GPDH activity. Muscle fibers in adult rat EOMs show unique metabolic properties not seen in other skeletal muscles, covering their extraordinary functional demands. It can be speculated that the EOMs embedded within the orbit material do not need to perform with high force, and therefore they could develop extensive systems that ensure both fatigue resistance (many mitochondria) and fast contraction with high mATPase activity (a well-developed sarcotubular system).

Perioculomotor cell groups in monkey and man defined by their histochemical and functional properties: Reappraisal of the Edinger‐Westphal nucleus

The perioculomotor region contains several functional cell groups, including parasympathetic preganglionic neurons of the ciliary ganglion, motoneurons of multiply innervated muscle fibers (MIF) of extraocular muscles, and urocortin-positive neurons. In this study, midbrain sections of monkey and human were treated with antibodies against choline acetyltransferase (ChAT), cytochrome oxidase (CytOx), nonphosphorylated neurofilaments (NP-NF), chondroitin sulfate proteoglycan (CSPG), and urocortin (UCN) to identify them by their histochemical properties. To facilitate the comparison between species, a new nomenclature was introduced (see also May et al., 2007), which designates these perioculomotor cell populations (pIII) in terms of their function and histochemical properties. The name Edinger-Westphal nucleus (EW) is kept for the cytoarchitecturally defined cell group traditionally considered as the location of preganglionic neurons of the ciliary ganglion. In monkey, the EW contains ChAT-positive presumed preganglionic neurons, and is therefore termed EW(PG), but in contrast human EW consists of noncholinergic UCN-positive neurons, and is therefore termed EW(U). In human, the presumed preganglionic neurons were found dorsal to EW(U), as an inconspicuous group of ChAT- and CytOx-positive neurons. They were interspersed with prominent CSPG-positive cells, a pattern also present in monkey. For the first time, the MIF motoneurons could be identified around the medial aspect of the human oculomotor nucleus as a group of ChAT-positive neurons that lack CSPG-positive perineuronal nets. Moreover, the Perlia nucleus was found to share the histochemical properties of oculomotor twitch motoneurons. The present results form the basis for addressing the appropriate functional cell groups in correlative clinicopathological studies.

Quantitative Proteomics Profiling of Sarcomere Associated Proteins in Limb and Extraocular Muscle Allotypes

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.

Determination of slow-tonic MyHC immunoreactivity is an important step in the evaluation of muscle spindles in porcine extraocular muscles

We have tested our hypothesis suggesting (i) that for the reliable determination and counting of muscle spindles (Msp) at the light microscopy level in extraocular muscles (EOM), analysis of the spindle specific myosin heavy chain (MyHC) immunoreactivity of intrafusal fibers, especially after staining with anti-slow-tonic MyHC antibodies, is the most convenient tool, (ii) that the number of Msp determined by the slow-tonic MyHC immunoreactivity of intrafusal fibers in EOM is much lower than that based on histological examination and (iii) that the previously reported numbers of Msp based on histological examination of EOM could be overestimated. In order to determine the number and distribution of Msp and to analyze the MyHC isoform immunoreactivity of intrafusal fibers in porcine EOM, paraffin sections of three 9-month-old pig medial (MR) and lateral rectus (LR), levator palpebrae (LP) and retractor bulbi (RB) muscles were stained histologically or using specific monoclonal antibodies (mAbs) against MyHC isoforms. Msp in recti and LP muscles studied by immunocytochemistry contained nuclear bag (NB) fiber(s) reacting with mAbs against slow-tonic, slow-twitch, α-cardiac and neonatal MyHCs, but not with the mAb against fast-twitch MyHC, which, on the contrary, stained nuclear chain (NC) fibers. Based on determination of spindle specific slow-tonic MyHC isoform immunoreactivity we have found 72 Msp in the MR and 68 Msp in the LR and 12 Msp in LP muscles, which was only 62, 55 and 32% of the Msp total counts according to histological examination, respectively. In the RB muscle, we have even found only 15 spindle-like-structures composed of encapsulated thin muscle fibers, which possessed only a reaction with anti-fast-twitch MyHC mAb, but lacked slow-tonic, slow-twitch or α-cardiac MyHCs immunoreactivity. Our analysis of porcine EOM confirmed the above suggestions, demonstrating, for the first time in the pig, the presence of “false Msp” mimicking encapsulated muscle fibers on histological sections that lack spindle specific MyHC immunoreactivity. In analogy with other muscles we suggest that “false Msp” are not innervated by sensory axons and therefore do not contribute to the physiological sensation of the muscle length changes. Our results thus show that the reliable identification of functionally effective Msp in EOM must involve immunohistochemical analysis of spindle specific MyHC isoforms of intrafusal fibers, as “false” spindles appearing on histologically stained sections as encapsulated muscle fibers could be regarded as “true” Msp and thus increase the spindle number counts in earlier studies.

Histochemical and immunohistochemical study on muscle fibers in human extraocular muscle spindles

Human extraocular muscles are unique in several ways including their endowment with proprioceptive organs. Aim of this study was to establish a classification of intrafusal muscle fibers of human extraocular muscles based on their histochemical and immunohistochemical properties and to determine their relationship to extrafusal extraocular muscle fiber types in this respect. Using light microscopy, intrafusal muscle fibers were followed on consecutive cross-sections and classified according to the localization of their myonuclei and to their enzyme- and myosin-immunohistochemical characteristics. Sixteen muscle spindles in human extraocular muscles counted as ‘true’ spindles revealed 27% nuclear chain fibers [40.1 μm ± 10.4; perimeter ± SD] and 73% anomalous fibers [44.1 μm ± 12]. Seven ‘false’ muscle spindles showed only anomalous fibers [43.8 μm ± 11.1] and entirely lacked nuclear chain fibers. Six fiber types were distinguished according to their histochemical and myosin heavy chain immunohistochemical properties. Fiber type 1 [46.3 μm ± 13.3] was made up of fast-twitch myosin heavy chain isoform. Fiber type 2 [39.5 μm ± 10] additionally expressed a developmental myosin heavy chain isoform. Fiber type 3 [42.8 μm ± 10.4] consisted of pure slow-twitch positive muscle fibers. Slow-twitch MHC and fast-twitch myosin heavy chain isoform were found in fiber type 4 [43.3 μm ± 9]. Fiber types 5 and 6 showed different myosin heavy chain patterns than fiber types 1–4. The vast majority of nuclear chain fibers displayed fiber type 2 features, but 12% of nuclear chain fibers were found to be of fiber type 1. Among anomalous fibers in true spindles the frequency of fiber type 1 was much higher than in false spindles. On the other hand, fiber type 4 was found more often in false than in true spindles. With regard to their histochemical and immunohistochemical properties intrafusal muscle fibers in human extraocular muscles differ both from intrafusal muscle fibers in other skeletal muscles and from extrafusal muscle fibers in extraocular eye muscles. These conspicuous differences to skeletal muscle spindles relate to their morphology and myosin heavy chain characteristics. In particular, the occurrence of anomalous fibers might reflect dynamic neuronal processes and might be necessary for modulating and adapting processes in advancing age, as well as maintaining proprioceptive input during the whole life.